Relative divergence times

For an identification of cophylogenetic events it is advantageous when relative ages of the two biont groups can be estimated. A plausible estimate for these relative ages might permit an appraisal of simultaneity in speciation events in associated taxa. Simultaneity of speciation events is a premise for the recognition of cospeciation events (Page & Charleston 1998) and allows a distinction between cospeciation events and coincidental congruencies in the phylogenies of associated taxa. As shown below, this differentiation will have an important impact on the evaluation of the evolutionary dependence of lichen bionts.

All Physciaceae are exclusively associated with Trebouxia (see chapter 4) and apart from clade S, all known Trebouxia clades appear compatible with the Physciaceae. It is therefore the most parsimonious assumption that Trebouxia is the ancestral photobiont of the Physciaceae. Since many other lichen families are also associated with Trebouxia, it might be assumed that the genus Trebouxia could be even older than the family Physciaceae. The mean p-distance found in Trebouxia was 0.16 and the maximal p-distance was 0.29 (I4c – G7b). The mean p-distance found in the Physciaceae was 0.18 and the maximal p-distance was 0.32 (Heterodermia leucomela – Buellia elegans / B. zoharyi). This discrepancy of assumed phylogenetic age and sequence divergence might be explained by lower mutation rates in Trebouxia than in the Physciaceae.

Therefore, in pairs of cospeciating taxa, the Trebouxia taxa should be separated by a lesser p-distance than the Physciacean sister species. The assumption of lower mutation rates in Trebouxia is concordant with the more general hypothesis that “inhabitants” might exhibit lower mutation rates than “exhabitants” (Law &

Lewis 1983). These authors argued that inhabitants are protected from environmental change by the exhabitants so that the former might experience a more stable environment than the latter. Environmental change, which forces organisms to adapt, was seen as the evolutionary pressure that promotes an increased mutation rate.

Searching for cospeciation events with TreeMap does not take divergence times into account. Therefore cospeciation events revealed by TreeMap had to be analyzed separately with respect to divergence times.

Only divergence times of cospeciation events were analyzed that included "terminal" mycobiont sister species. This was done in order to minimize the uncertainty caused by deviating mutation rates in the associated organisms. Analyzing p-distances in Physciacean sister species and their associated Trebouxia photobionts revealed just the opposite of the above assumption about the relation of p-distances in coevolving pairs of sister taxa. In all incidences of potential cospeciation events, the Trebouxia taxa were separated by larger genetic distances than the mycobionts (TABLE 5.6, FIG.5.4). In many of these instances

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia

the photobiont p-distance was much larger, indicating that these unequal divergences could not be explained by deviating mutation rates. Therefore, these taxa speciated at different times. This allowed the rejection of the cospeciation hypothesis in most if not all analyzed instances. These findings revealed a pronounced contrast between the apparent ecological dependence of most mycobionts from their photobionts as concluded from the commonly observed high degree of selectivity and the evolutionary independence of the two biont groups as demonstrated by the lack of any significant degree of congruence between the two biont phylogenies.

TABLE 5.5: Number of cospeciation events that could be expected from chance alone, as tested by randomizing host (Trebouxia) and associate (Physciaceae) trees. Ø: average number of cospeciation events, P<5%: minimal number of cospeciation events that had a probability smaller than 5%. Nineteen cospeciation events were detected by TreeMap in the original data.

Ø P < 5%

1000 random photobiont trees 12 16 or more 4000 random mycobiont trees 20 25 or more 5000 randomizations of both biont’s trees 17 22 or more

FIG. 5.3: Probabilities (%) of different numbers of cospeciation events occuring just by chance alone.

Histograms produced with TreeMap in randomisation tests of A) photobiont phylogeny (1000 randomisations),

B) mycobiont phylogeny (4000 randomisations), or C) both biont’s phylogenies (5000 randomisations).

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia

Trebouxia ITS phylogeny Physciaceae ITS phylogeny

F^IG.5.4: with the same subclade or one species was associated with multiple photobiont subclades that "enclosed"

the subclade of its sister species. Lines connecting

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia

TABLE 5.6: Comparison of p-distances (Full-length sequences were used in p-distance estimation) between mycobiont sister taxa and their compatible photobionts. These potential cospeciation events are illustrated in FIG. 5.4. Ana:

Anaptychia, Der: Dermatiscum, Dim: Dimelaena, Dip: Diplotomma, Dir: Dirinaria, Pco: Physconia, Pha:

Phaeophyscia, Pia: Physcia, Pyx: Pyxine, Rin: Rinodina.

node paired pairs p-distance (%)

Trebouxia

p-distance (%) Physciaceae

a G3 / G8 – Pyx. farinosa / Pyx. sorediata 24 13

b G3 / (G7/9) – Dir. sp. 1 / Dir. applanata 21 / 16 13

c A2 / (A5/9) – Dip. lutosum / Dip. venustum 10 / 8 6

d G5 / I1 – Der. thunbergii / Dim. oreina 20 15

e A2 / A1 – Rin. gennarii / Rin. oleae 6 3

f G4 / (G7 / G9) – Pia. krogiae / (Pia. undulata / Pia. atrostriata) 21 / 17 11

g G7 / G9 – Pia. undulata / Pia. atrostriata 16 6

h A2 / I1 – Rin. obnascens / Rin. milvina 17 3

i A4 / I1 – Rin. luridescens / Rin. lecanorina 16 6

j I3 / I1 – Pha. endophoenicea / Pha. orbicularis 7 6

k A3 / A1 – Ana. ulotrichoides / Ana. ciliaris 6 6

l A4 / I1 – Ana. runcinata / Pco. grisea 16 6

Most Physciaceae species appeared to be selective at the level of Trebouxia subclades. But not only species were found to be selective, even supraspecific taxa showed a remarkable constancy in photobiont choice.

Nine mycobiont lineages (a – i in FIG. 5.5) appeared to be selective for particular Trebouxia subclades. From the 77 species included in this analysis, 50 species were included in these nine mycobiont lineages. In other words, the majority of speciation events in the Physciaceae was not triggered by speciation events in Trebouxia. Twenty-eight duplication events could be counted in the mycobiont phylogeny and are therefore considered to be a common event in Physciaceae evolution. Interestingly, 14 species of these nine lineages switched their photobiont (FIG. 5.5). The phylogenetic steadiness of mycobiont choice on one side (as demonstrated by the frequency of duplication events), the frequent number of alga switches on the other side, as well as the lack of cospeciation events suggests that factors other than mycobiont selectivity determine the evolution of biont association.

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia

Trebouxia ITS phylogeny Physciaceae ITS phylogeny

F^IG.5.5: Duplications and species. Forty of these 50 species appeared photobiont steady referring to 34 duplication events (blue dots, bold lines). Fourteen of the 50 species could be assigned to host switches

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia